3145
J. Am. Chem. SOC.1992,114, 3 145-3 147 cross-linked DNAs9 The desired bands were cut from the gel, isolated by electroelution, treated with piperidine (90 OC, 30 min), and resubmitted to PAGE analysis. Cross-linked material 5’end-labeled on the shorter strand afforded a major cut at the guanine at position 12 (Glz) when compared to a Maxam and Gilbert G-specific laneelo Likewise, cross-linked material 5’end-labeled on the longer strand afforded cleavage at the AI7 residue, corresponding to a three base pair bisalkylation between G and A residues in the sequence s-GCT-3’ 3’-CGA-5’
Conversion of isolated cross-link to a major cleavage site on each strand suggests that the covalent bond formation occurs at the piperidine-labile locations. 11,12 Inosine (I) and the modified nucleoside 7-deazaguanine (Z) were synthetically incorporated to evaluate potential base alkylation sites. Substitution of the G I 2 residue involved in the G-to-A cross-link in 1 with 7-deazaguanine 2 resulted in no cross-link formation. No piperidine-sensitive scission was detected at this base even in single-stranded experiments. Cross-links were generated upon replacement of GI8with 7-deazaguanine 3, but were not observed with either T (6) or A (7) substitution at this base. Substitution of inosine for either the Glz or GIs residue afforded cross-links (4, 5). However, yields were lower for 4, consistent with the decreased nucleophilicity of the N7 atom of inosine.13 Substitution of the A17 residue with G afforded G-to-G bisalkylations in the sequences 5’-GCC-3’ 3’-CGG-5’
5’-GAC-3’
3’-CTG-5’
8
9
Attempts to induce G-to-G cross-links involving adjacent base pairs failed (10).14 All G residues in either single strand of 1 are alkylated by carzinophilin at various levels of efficiency, whereas all A residues (including A17) are inert (data not shown). Given the differences in nucleophilicity of N7 of A and G, these results support a model for cross-link formation in which the first alkylation at GI2directs the second alkylation a t A17 (Figure 1). The reaction at the usually inert A17 residue is thus template directed, and the kinetics (9) DNA-CZ complexes were treated with 0.2 N NaOH in order to prevent depurination of cross-links when submitted to PAGE analysis. Cross-linked DNA which had not been exposed to base (obtained in low yield) could be electroeluted from gel and converted to material of identical migration to that which had been treated with NaOH prior to gel analysis. Stabilization of N7 alkylations under similar conditions has been previously reported: Kohn, K. W.; Spears, C. L. Biochim. Biophys. Acta 1%7,145,743. For a recent demonstration of nitrogen mustard cross-link stabilization using base to elucidate the novel 5’-GXC-3’ sequence selectivity of mechloroethylenamine, see: Ojwang, J. 0.;Grueneberg, D. A.; Loechler, E. L. Cancer Res. 1989, 6529. (10) Maxam, A. M.; Gilbert, W. Merhods Enzymol. 1980, 65, 499. (1 1) Alkylations (
